Ferroptosis is a regulated form of lipotoxic cell death that involves iron-dependent generation of reactive oxygen species (ROS) and the accumulation of oxidatively damaged lipids (e.g. lipid peroxides). Ferroptosis has been implicated in the etiology of degenerative diseases, such as neurodegeneration associated with iron accumulation. Cells contain a protective pathway in which the glutathione-dependent peroxidase GPX4 repairs lipid peroxides and blocks cell death. Targeted induction of ferroptosis by inhibiting GPX4 has proven to be an efficacious treatment in in vitro and in vivo models of cancer, including drug-resistant forms of cancer. Despite the excitement from these recent findings, our understanding of the mechanisms underlying ferroptosis remains limited. Furthermore, many cancer cells are resistant to ferroptosis and the mechanisms of ferroptosis resistance in cancer remains mostly unknown. To overcome this critical gap in knowledge, we performed a synthetic lethal, whole-genome CRISPR screen to identify factors that protect cancer cells from ferroptosis. Our findings identify the lipid droplet oxidoreductase AIFM2 as a key factor that promotes ferroptosis resistance in cancer. Deletion of AIFM2 dramatically sensitizes cells to ferroptosis and AIFM2 levels correlate with cancer resistance across hundreds of cancer lines, indicating that AIFM2 is a biomarker of ferroptosis resistance and suggesting that it is broadly involved in ferroptosis resistance across many types of cancer. Our proposed research builds on our discovery and employs a combination of functional genomic, cell biology, and biochemistry strategies to achieve the following goals: 1) elucidate the mechanism by which AIFM2 prevents lipid damage and ferroptosis, 2) define the relationship between lipid droplets, fatty acid metabolism, and ferroptosis, and 3) identify new factors involved in protecting cancer cells from ferroptosis. These goals are potentially transformative because they focus on new mechanisms of ferroptosis resistance in cancer cells that act in parallel to the canonical glutathione-based protective system.